Degeneration of dopaminergic neurons induced by thrombin injection in the substantia nigra of the rat is enhanced by dexamethasone: role of monoamine oxidase enzyme.

Anti-inflammatory strategies receive growing attention for their potential to prevent pathological deterioration in disorders such as Parkinson's disease, which is accompanied by inflammatory reactions that might play a critical role in the degeneration of nigral dopaminergic neurons. We investigated the influence of dexamethasone - a potent synthetic member of the glucocorticoids class of steroid hormones that acts as an anti-inflammatory - on the degeneration of the dopaminergic neurons of rats observed after intranigral injection of thrombin, a serine protease that induces inflammation through microglia proliferation and activation. We evaluated tyrosine hydroxylase (TH)-positive neurons as well as astroglial and microglial populations; dexamethasone prevented the loss of astrocytes but was unable to stop microglial proliferation induced by thrombin. Moreover, dexamethasone produced alterations in the levels of nexin and the thrombin receptor PAR-1, and facilitated accumulation of alpha-synuclein induced by thrombin in dopaminergic neurons. Dexamethasone increased oxidative stress and expression of monoamine oxidase A and B, along with changes on different MAP kinases related to degenerative processes, resulting in a bigger loss of dopaminergic neurons after intranigral injection of thrombin in dexamethasone-treated animals. It is interesting to ascertain that inhibition of monoamine oxidase by tranylcypromine prevented neurodegeneration of dopaminergic neurons, thus suggesting that the deleterious effects of dexamethasone might be mediated by monoamine oxidase.

The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination.

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining - a non-specific neuronal marker - and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of alpha-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.

Thrombin induces in vivo degeneration of nigral dopaminergic neurones along with the activation of microglia.

Seven days after the injection of different concentrations of thrombin into the nigrostriatal pathway, a strong macrophage/microglial reaction was observed in the substantia nigra (SN), indicated by immunostaining, using OX-42 and OX-6 antibodies, and by the induction of iNOS, IL-1alpha, Il-1beta and TNF-alpha. Moreover, selective damage to dopaminergic neurones was produced after thrombin injection, evidenced by loss of tyrosine hydroxylase immunostaining and tyrosine hydroxylase mRNA-expressing cell bodies, and the unaltered transcription of glutamic acid decarboxylase mRNA in the SN and striatum. These thrombin effects could be produced by its ability to induce the activation of microglia described in in vitro studies, and are in agreement with the effects described for other proinflammatory compounds. Thrombin effects are produced by its biological activity since they almost disappeared when thrombin was heat-inactivated or injected along with its inhibitor alpha-NAPAP. Thrombin is a multi-functional serine protease rapidly produced from prothrombin at the sites of tissue injury, and also upon breakdown of the blood-brain barrier, which strongly suggests it could easily enter into the CNS. These results could have special importance in some degenerative processes of the nigrostriatal dopaminergic system.